Method for expression of small RNA molecules within a cell

ABSTRACT

The invention provides methods and compositions for the expression of small RNA molecules within a cell using a lentiviral vector. The methods can be used to express doubles stranded RNA complexes. Small interfering RNA (siRNA) can be expressed using the methods of the invention within a cell, which are capable of down regulating the expression of a target gene through RNA interference. A variety of cells can be treated according to the methods of the invention including embryos, embryogenic stem cells, allowing for the generation of transgenic animals or animals constituted partly by the transduced cells that have a specific gene or a group of genes down regulated.

REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority under 35 U.S.C. §119(e)to U.S. Provisional Application No. 60/322,031, filed Sep. 13, 2001,U.S. Provisional Application No. 60/347,782, filed Jan. 9, 2002, U.S.Provisional Application No. 60/389,592, filed Jun. 18, 2002, and U.S.Provisional Application No. , attorney docket number CALTE.011PR, filedAug. 27, 2002.

GOVERNMENT SUPPORT

[0002] This invention was made with government support under GrantNumber GM39458 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates generally to methods for alteringgene expression in a cell or an animal using viral constructs engineeredto deliver an RNA molecule. In a more specific aspect, a viral constructis used to deliver double-stranded RNA molecules that can be used todown-regulate or modulate gene expression.

[0005] 2. Description of the Related Art

[0006] RNA interference (RNAi) or silencing is a recently discoveredphenomenon (A. Fire et al., Nature 391, 806 (1998); C. E. Rocheleau etal. Cell 90, 707 (1997)). Small interfering RNAs (“siRNAs”) aredouble-stranded RNA molecules that inhibit the expression of a gene withwhich they share homology. siRNAs have been used as a tool to downregulate the expression of specific genes in a variety of cultured cellsas well as in invertebrate animals. A number of such approaches havebeen reviewed recently (P. D. Zamore Science 296, 1265 (2002)); however,such approaches have limitations. For example, no technique prior to theinvention described herein allows for the generation of transgenicmammal having a specific gene down regulated through RNA interference.Similarly, there is a need for more robust methods for the introductionof small RNA molecules with regulatory function. The invention providedherein addresses these and other limitations in the field of RNAmediated gene regulation.

SUMMARY OF THE INVENTION

[0007] The invention relates generally to methods to express within acell an RNA molecule or molecules. These methods can be used with a widevariety of cell types. RNA molecules can be expressed within a cell fora variety of purposes. For example, and without limitation, RNAmolecules can serve as markers within a cell, can act as antisenseoligonucleotides or ribozymes for regulating gene expression, and canserve to down regulate genes through RNA interference.

[0008] In one aspect, the invention provides retroviral constructs forthe expression of an RNA molecule or molecules within a cell. Theconstructs preferably comprise a nucleic acid having the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR), aself-inactivating lentiviral 3′ LTR, and a RNA Polymerase III (pol III)promoter. The retroviral constructs preferably comprise an RNA codingregion operably linked to the RNA Polymerase III promoter. The RNAcoding region preferably comprises a DNA sequence that can serve as atemplate for the expression of a desired RNA molecule.

[0009] The RNA coding region can be immediately followed by a pol IIIterminator sequence which directs the accurate and efficient terminationof RNA synthesis by pol III. The pol III terminator sequences generallycomprise 4 or more consecutive T residues. In a preferred embodiment, acluster of 5 consecutive Ts is used as the terminator by which pol IIItranscription is stopped at second or third T of the DNA template. As aresult, only 2 to 3 U residues are added to the 3′ end of the RNA thatis synthesized from the RNA coding region.

[0010] A variety of pol III promoters can be used with the invention,including for example, the promoter fragments derived from H1 RNA genesor U6 sn RNA genes of human or mouse origin or from any other species.In addition, pol III promoters can be modified/engineered to incorporateother desirable properties such as to be inducible by small chemicalmolecules either ubiquitously or in a tissue-specific manner, forexample, one activated with tetracycline or IPTG (lacI system).

[0011] The pol III promoter, RNA template region and pol III terminatortogether may comprise an “RNA cassette” or “RNA expression cassette.” Ifthe RNA is a small inhibitory RNA (siRNA), the expression cassette maybe termed an “siRNA expression cassette.”

[0012] In one embodiment, the RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region. Such an RNA molecule when expressed desirablyforms a “hairpin” structure. The loop region is generally between about2 and about 10 nucleotides in length. In a preferred embodiment, theloop region is from about 6 and about 9 nucleotides in length. In onesuch embodiment of the invention, the sense region and the antisenseregion are between about 15 and about 30 nucleotides in length.

[0013] In one embodiment, the RNA coding region is operably linkeddownstream to an RNA Polymerase III promoter such that the RNA codingsequence can be precisely expressed without any extra non-codingnucleotides present at 5′ end. In this way an RNA sequence can beexpressed that is identical to a target sequence at the 5′ end. Thesynthesis of the RNA coding region is ended at the terminator site. Inone preferred embodiment the terminator consists of five consecutive Tresidues.

[0014] In another aspect of the invention, the retroviral vector cancomprise multiple RNA coding regions. In one embodiment, the retroviralconstruct comprises a first RNA pol III promoter, a first coding regionencoding a first RNA molecule operably linked to the first RNA pol IIIpromoter, a second RNA pol III promoter and a second RNA coding regionoperably linked to the second RNA pol III promoter. Preferably, thesecond RNA coding region encodes an RNA molecule that is substantiallycomplementary to the RNA molecule encoded by the first RNA codingregion, such that the two RNA molecules can form a double-strandedstructure when expressed. The methods of invention also include multipleRNA coding regions that encode hairpin-like self-complementary RNAmolecules or other non-hairpin molecules.

[0015] In yet another embodiment of the invention, the retroviralconstruct comprises a first RNA pol III promoter operably linked to afirst RNA coding region, and a second RNA pol III promoter operablylinked to the same first RNA coding region in the opposite direction,such that expression of the RNA coding region from the first RNA pol IIIpromoter results in a synthesis of a first RNA molecule as the sensestrand and expression of the RNA coding region from the second RNA polIII promoter results in synthesis of a second RNA molecule as anantisense strand that is substantially complementary to the first RNAmolecule. In one such embodiment, both RNA Polymerase III promoters areseparated from the RNA coding region by termination sequences,preferably termination sequences having five consecutive T residues.

[0016] According to one embodiment of the invention, the 5′ LTRsequences in the retroviral construct are derived from HIV. Theretroviral construct may also comprise a woodchuck hepatitis virusenhancer element sequence and/or a tRNA amber suppressor sequence.

[0017] In another embodiment of the invention, the self-inactivating 3′LTR is a U3 element with a deletion of its enhancer sequence. In yetanother embodiment, the self-inactivating 3′ LTR is a modified HIV 3′LTR.

[0018] The recombinant retroviral construct can be pseudotyped, forexample with the vesicular stomatitits virus envelope glycoprotein.

[0019] In another aspect of the invention, expression of the RNA codingregion results in the down regulation of a target gene. Preferably thetarget gene comprises a sequence that is at least about 90% identicalwith the RNA coding region, more preferably at least about 95%identical, and even more preferably at least about 99% identical.

[0020] According to a further aspect of the invention, the viralconstruct also comprises a nucleotide sequence encoding a gene ofinterest. The gene of interest is preferably operably linked to aPolymerase II promoter. Such a construct also can contain, for example,an enhancer sequence operably linked with the Polymerase II promoter.

[0021] A variety of Polymerase II promoters can be used with theinvention, including for example, the CMV promoter. The RNA PolymeraseII promoter that is chosen can be a ubiquitous promoter, capable ofdriving expression in most tissues, for example, the human Ubiquitin-Cpromoter, CMV β-actin promoter or PGK promoter. In other embodiments theRNA Polymerase II promoter is a tissue-specific promoter.

[0022] In one embodiment, the gene of interest is a marker or reportergene, that can be used to verify that the vector was successfullytransfected or transduced and its sequences expressed. In one suchembodiment, the gene of interest is a fluorescent reporter gene, forexample, the Green Fluorescent Protein. In yet another embodiment, thegene of interest is a drug resistant gene which can be used to selectthe cells that are successfully transduced. For example, the drugresistant gene can be the zeocin resistant gene (zeo). The gene ofinterest also can be a hybrid of a drug resistant gene and a fluorescentreporter gene, such as a zeo/gfp fusion. In another embodiment, the geneof interest encodes a protein factor that can regulate the transcriptionactivity of inducible pol III promoters. In one of such embodiment, thegene of interest is tetR (repressor for tet operon) which regulatestetracycline responsive pol III promoters.

[0023] It is another aspect of the invention to provide methods forexpressing an RNA molecule or molecules within a cell. According to theinvention, a packaging cell line is transfected with a retroviralconstruct of the invention, recombinant retroviral particles arerecovered from the packaging cell line; and a target cell is infectedwith the recombinant retrovirus particles.

[0024] In one embodiment of the invention, the target cell is anembryonic cell. An embryonic cell may be, for example, a single cellembryo or embryonic cells from within an early-stage embryo. In anotherembodiment of the invention, the target cell is an embryogenic stemcell. When the target cell is an embryonic cell, in one embodiment theembryonic cell is infected by injecting the recombinant retrovirusbetween the zona pellucida and the cell membrane of the embryonic cell.In another embodiment, the embryonic cell is infected by removing thezona pellucida and incubating the cell in solution containing therecombinant retrovirus. In such an embodiment, the zona pellucida can beremoved, for example, by enzymatic digestion.

[0025] When the target cell is an embryonic cell or an embryogenic stemcell, the cell may be transplanted in a pseudopregnant female togenerate a transgenic animal.

[0026] The methods of the invention also can be used with a variety ofprimary ex vivo normal or diseased cells or cells adapted in varioustissue culture conditions from human, mouse and other vertebrates,including, without limitation, stem or precursor cells for thehematopoictic system, central nerve system cells, cells withregenerative capacities from a variety of other tissues and organs,dendritic cells and other developing and mature myeloid and lymphoidcells, and cancer cells derived from different cell lineages.

[0027] In a particular embodiment, the target cell is an embryonic cellof a bird within an egg. The embryonic cell of a bird is preferablyinfected by contacting the embryonic blastodisc of the bird egg withretroviral particles.

[0028] In yet another embodiment, the target cell is a fish egg. Thefish egg is preferably infected by delivering the retroviral particlesto the space between the chorion and the cell membrane of the fish egg.

BRIEF DESCRIPTION OF THE DRAWINGS

[0029]FIG. 1A shows a schematic diagram of a retroviral vector carryingan expression cassette for RNA expression, termed “RNA cassette” and a“Marker Gene” or gene of interest. The RNA expression cassette can beembedded at any permissible sites of the retroviral construct either assingle copy or multiple tandem copies. In addition, although notindicated in the figure, more than one RNA expression cassette may bepresent in the retroviral construct. FIG. 1B shows a similar constructin which the RNA expression cassettes flank a marker gene.

[0030]FIG. 2 shows a schematic view of an RNA expression cassettecomprising an RNA Polymerase III promoter 100 linked to an RNA codingregion 110-130 and a terminator sequence 140. The RNA coding regioncomprises a sense region 110, a loop region 120, and an antisense region130,

[0031]FIG. 3 shows a schematic view of an RNA expression cassette havingan RNA Polymerase III promoter 100 linked to a first RNA coding region110 and a first terminator sequence 140 and a second RNA polymerase IIIpromoter 105 linked to a second RNA coding region 115 and a secondterminator 145.

[0032]FIG. 4 shows a schematic view of an RNA expression cassette havinga first RNA Polymerase III promoter 100 linked to an RNA coding region110 and a first terminator sequence 145. The expression cassette has asecond RNA polymerase III promoter 105 linked to the RNA coding region115, the same sequence as 110 in reverse, and a second terminator 140.

[0033]FIG. 5. Schematic illustration of a lacZ siRNA encoding lentiviralvector. 5′LTR: an HIV based lentiviral vector 5′ LTR; F: an HIV Flapelement; pol III: a human H1-RNA pol III promoter (−240 to −8); siRNA: alacZ specific small hairpin RNA coding region and its structure anddetailed sequence are illustrated below. UbiC: an internal humanubiquitinC promoter; GFP: a GFP marker gene driven by UbiC promoter. W:a woodchuck RNA regulatory element. 3′LTR: an HIV based selfinactivating lentiviral 3′ LTR.

[0034]FIG. 6. A lacZ specific siRNA encoded by a lentiviral vector canefficiently inhibit the expression of lacZ reporter gene in virustransduced mammalian cells. MEF: mouse embryonic fibroblasts; HEK293:human embryonic kidney cells. Both of the test cell lines harbor lacZand firefly luciferase reporter genes, and the expression levels of thereporter genes can be measured by chemiluminescent assays. Ctrl: theratio of lacZ activity versus Luc activity of the uninfected parentalcells, which was arbitrarily set to 1. Transduced: the specificinhibition of lacZ expression calculated as the reduction of lacZ to Lucratio.

[0035]FIG. 7. Transgenic animals that express a lacZ specific siRNAmolecule encoded by a lentiviral vector can successfully suppress theexpression of the ubiquitous lacZ reporter gene in a ROSA26+/−background. ROSA1-6: the lacZ activities in the limb tissues of sixE17.5 ROSA26+/− embryos which served as positive controls. Thedifference in lacZ activity between individual ROSA26+/− embryos mayresult from variable protein extraction efficiency. TG1-4: the lacZactivities in the limb tissues of four E17.5 transgenic embryosexpressing a lentiviral vector-encoded lacZ siRNA molecule in ROSA+/−background. WT1-6: lacZ activity in the limb tissues of six E17.5C57B1/6 wildtype embryos, included as the negative control. Thebackground levels of endogenous beta-galactosidase activity are generalbelow 1,000 LU/ug, thus the columns are not visible.

[0036]FIG. 8 shows a schematic illustration of a Tet-inducible lacZsiRNA lentiviral vector. A Tet repressor gene (TetR; SEQ ID NO: 5) isthe under the control human UbiquitinC promoter and its expression canbe monitored by the downstream GFP marker coupled by IRES element(internal ribosomal entry site). The anti-lacZ siRNA cassette is drivenby a Tet-inducible pol III promoter derived from human U6-promoter (−328to +1) containing a single TetR binding site (TetO1) between the PSE andTATA box (SEQ ID NO: 4). In the absence of tetracycline, TetR binds tothe promoter and its expression is repressed. Upon the addition oftetracycline, TetR is moved from the promoter and transcription starts.

[0037]FIG. 9 shows the results of an experiment that demonstrated that aTet-inducible siRNA expression cassette can regulate gene expression inresponse to Doxycycline treatment. lacZ and luciferase double expressingHEK293 cells (293Z+Luc) were transduced with a lentiviral vectorcarrying a Tet-inducible lacZ-siRNA cassette and a Tet repressor underthe control of a UbiquitinC promoter (FIG. 8). The transduced cells weretreated with 10 ug/ml Doxycycline (Plus Dox) for 48 hr or without theDoxycycline treatment as a control (No Dox). LacZ and luciferaseactivities were measured as described in the previous figures. Therelative suppression activity is calculated as the ratio of lacZ versusluciferase and No Dox control was arbitrarily set to 1.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENT

[0038] The inventors have previously identified a method for introducinga transgene of interest into a cell or animal. This technique isdescribed in co-pending U.S. provisional patent application No.60/322,031 filed on Sep. 13, 2001 and co-pending U.S. provisional patentapplication No. 60/347,782 filed on Jan. 9, 2002, the entire contents ofwhich are incorporated herein by reference.

[0039] Unless defined otherwise, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which this invention belongs. Any methods, devicesand materials similar or equivalent to those described herein can beused in the practice of this invention.

[0040] By “transgene” is meant any nucleotide sequence, particularly aDNA sequence, that is integrated into one or more chromosomes of a hostcell by human intervention, such as by the methods of the presentinvention. In one embodiment, a transgene is an “RNA coding region.” Inanother embodiment the transgene comprises a “gene of interest.” Inother embodiments the transgene can be a nucleotide sequence, preferablya DNA sequence, that is used to mark the chromosome where it hasintegrated. In this situation, the transgene does not have to comprise agene that encodes a protein that can be expressed.

[0041] A “gene of interest” is a nucleic acid sequence that encodes aprotein or other molecule that is desirable for integration in a hostcell. In one embodiment, the gene of interest encodes a protein or othermolecule the expression of which is desired in the host cell. In thisembodiment, the gene of interest is generally operatively linked toother sequences that are useful for obtaining the desired expression ofthe gene of interest, such as transcriptional regulatory sequences.

[0042] A “functional relationship” and “operably linked” mean, withoutlimitation, that the gene is in the correct location and orientationwith respect to the promoter and/or enhancer that expression of the genewill be affected when the promoter and/or enhancer is contacted with theappropriate molecules.

[0043] An “RNA coding region” is a nucleic acid that can serve as atemplate for the synthesis of an RNA molecule, such as an siRNA.Preferably, the RNA coding region is a DNA sequence.

[0044] A “small interfering RNA” or “siRNA” is a double-stranded RNAmolecule that is capable of inhibiting the expression of a gene withwhich it shares homology. In one embodiment the siRNA may be a “hairpin”or stem-loop RNA molecule, comprising a sense region, a loop region andan antisense region complementary to the sense region. In otherembodiments the siRNA comprises two distinct RNA molecules that arenon-covalently associated to form a duplex.

[0045] The term “transgenic” is used herein to describe the property ofharboring a transgene. For instance, a “transgenic organism” is anyanimal, including mammals, fish, birds and amphibians, in which one ormore of the cells of the animal contain nucleic acid introduced by wayof human intervention, such as by the methods described herein. In atransgenic animal that comprises a transgene that encodes a gene ofinterest, the transgene typically causes the cell to express oroverexpress a recombinant protein. However, according to the methods ofthe invention, expression of an RNA coding region can be used to downregulate the expression of a particular gene through antisense or RNAinterference mechanisms.

[0046] The terms “founder,” “founder animal” and “founder line” refer tothose animals that are mature products of the embryos or oocytes towhich the transgene was added, i.e. those animals that grew from theembryos or oocytes into which DNA was inserted.

[0047] The terms “progeny” and “progeny of the transgenic animal” referto any and all offspring of every generation subsequent to theoriginally transformed animal.

[0048] The term “animal” is used in its broadest sense and refers to allanimals including mammals, birds, fish, reptiles and amphibians.

[0049] The term “mammal” refers to all members of the class Mammalia andincludes any animal classified as a mammal, including humans, domesticand farm animals, and zoo, sports or pet animals, such as mouse, rabbit,pig, sheep, goat, cattle and higher primates.

[0050] The term “oocyte” refers to a female gamete cell and includesprimary oocytes, secondary oocytes and mature, unfertilized ovum. Asused herein, the term “egg” when used in reference to a mammalian egg,means an oocyte surrounded by a zona pellucida. The term “zygote” refersto a fertilized ovum. The term “embryo” broadly refers to an animal inthe early stages of development.

[0051] “Perivitelline space” refers to the space located between thezona pellucida and the cell membrane of a mammalian egg or embryoniccell.

[0052] “Target cell” or “host cell” means a cell that is to betransformed using the methods and compositions of the invention.

[0053] “Lentivirus” refers to a genus of retroviruses that are capableof infecting dividing and non-dividing cells. Several examples oflentiviruses include HIV (human immunodeficiency virus; including HIVtype 1, and HIV type 2), the etiologic agent of the human acquiredimmunodeficiency syndrome (AIDS); visna-maedi, which causes encephalitis(visna) or pneumonia (maedi) in sheep, the caprinearthritis-encephalitis virus, which causes immune deficiency, arthritis,and encephalopathy in goats; equine infectious anemia virus, whichcauses autoimmune hemolytic anemia, and encephalopathy in horses; felineimmunodeficiency virus (FIV), which causes immune deficiency in cats;bovine immune deficiency virus (BIV), which causes lymphadenopathy,lymphocytosis, and possibly central nervous system infection in cattle;and simian immunodeficiency virus (SIV), which cause immune deficiencyand encephalopathy in sub-human primates.

[0054] A lentiviral genome is generally organized into a 5′ longterminal repeat (LTR), the gag gene, the pol gene, the env gene, theaccessory genes (nef, vif, vpr, vpu) and a 3′ LTR. The viral LTR isdivided into three regions called U3, R and U5. The U3 region containsthe enhancer and promoter elements. The U5 region contains thepolyadenylation signals. The R (repeat) region separates the U3 and U5regions and transcribed sequences of the R region appear at both the 5′and 3′ ends of the viral RNA. See, for example, “RNA Viruses: APractical Approach” (Alan J. Cann, Ed., Oxford University Press,(2000)), O Narayan and Clements J. Gen. Virology 70:1617-1639 (1989),Fields et al. Fundamental Virology Raven Press. (1990), Miyoshi H,Blomer U, Takahashi M, Gage F H, Verna I M. J Virol. 72(10):8150-7(1998), and U.S. Pat. No. 6,013,516.

[0055] Lentiviral vectors are known in the art, including several thathave been used to transfect hematopoietic stem cells. Such vectors canbe found, for example, in the following publications, which areincorporated herein by reference: Evans J T et al. Hum Gene Ther1999;10:1479-1489; Case S S, Price M A, Jordan C T et al. Proc Natl AcadSci USA 1999;96:2988-2993; Uchida N, Sutton R E, Friera A M et al. ProcNatl Acad Sci USA 1998;95:11939-11944; Miyoshi H, Smith K A, Mosier D Eet al. Science 1999;283:682-686; Sutton R E, Wu H T, Rigg R et al. Humanimmunodeficiency virus type 1 vectors efficiently transduce humanhematopoietic stem cells. J Virol 1998;72:5781-5788.

[0056] “Virion,” “viral particle” and “retroviral particle” are usedherein to refer to a single virus comprising an RNA genome, pol genederived proteins, gag gene derived proteins and a lipid bilayerdisplaying an envelope (glyco)protein. The RNA genome is usually arecombinant RNA genome and thus may contain an RNA sequence that isexogenous to the native viral genome. The RNA genome may also comprise adefective endogenous viral sequence.

[0057] A “pseudotyped” retrovirus is a retroviral particle having anenvelope protein that is from a virus other than the virus from whichthe RNA genome is derived. The envelope protein may be from a differentretrovirus or from a non-retroviral virus. A preferred envelope proteinis the vesicular stomatitius virus G (VSV G) protein. However, toeliminate the possibility of human infection, viruses can alternativelybe pseudotyped with ecotropic envelope protein that limit infection to aspecific species, such as mice or birds. For example, in one embodiment,a mutant ecotropic envelope protein is used, such as the ecotropicenvelope protein 4.17 (Powell et al. Nature Biotechnology18(12):1279-1282 (2000)).

[0058] The term “provirus” is used to refer to a duplex DNA sequencepresent in a eukaryotic chromosome that corresponds to the genome of anRNA retrovirus. The provirus may be transmitted from one cell generationto the next without causing lysis or destruction of the host cell.

[0059] A “self-inactivating 3′ LTR” is a 3′ long terminal repeat (LTR)that contains a mutation, substitution or deletion that prevents the LTRsequences from driving expression of a downstream gene. A copy of the U3region from the 3′ LTR acts as a template for the generation of bothLTR's in the integrated provirus. Thus, when the 3′ LTR with aninactivating deletion or mutation integrates as the 5′ LTR of theprovirus, no transcription from the 5′ LTR is possible. This eliminatescompetition between the viral enhancer/promoter and any internalenhancer/promoter. Self-inactivating 3′ LTRs are described, for example,in Zufferey et al. J. Virol. 72:9873-9880 (1998), Miyoshi et al. J.Virol. 72:8150-8157 and Iwakuma et al. Virology 261:120-132 (1999).

[0060] The term “RNA interference or silencing” is broadly defined andincludes all posttranscriptional and transcriptional mechanisms of RNAmediated inhibition of gene expression, such as those described in (P.D. Zamore Science 296, 1265 (2002)).

[0061] In one aspect of the invention, a recombinant retrovirus is usedto deliver a transgene comprising an RNA coding region of interest to atarget cell. Preferably the target cell is a mammalian cell. The cellmay be a primary cell, or may be a cultured cell, for example an withoutlimitation an HEK, CHO, COS, MEF, 293 cell. In one embodiment the targetcell is an oocyte or an embryonic cell, more preferably a one-cellembryo. The RNA coding region and any associated genetic elements arethus integrated into the genome of the target cell as a provirus. Whenthe target cell is an embryo, the cell may then be allowed to developinto a transgenic animal by methods well known in the art.

[0062] The recombinant retrovirus used to deliver the RNA coding regionis preferably a modified lentivirus, and thus is able to infect bothdividing and non-dividing cells. The recombinant retrovirus preferablycomprises a modified lentiviral genome that includes the transgene.Further, the modified lentiviral genome preferably lacks endogenousgenes for proteins required for viral replication, thus preventingundesired replication, such as replication in a resulting transgenicanimal. The required proteins are preferably provided in trans in thepackaging cell line during production of the recombinant retrovirus, asdescribed below.

[0063] In another embodiment, the recombinant retrovirus used to deliverthe RNA coding region is a modified Moloney virus, for example a MoloneyMurine Leukemia Virus. In a further embodiment, the virus is a MurineStem Cell Virus (Hawley, R. G., et al. (1996) Proc. Natl. Acad. Sci. USA93:10297-10302; Keller, G., et al. (1998) Blood 92:877-887; Hawley, R.G., et al. (1994) Gene Ther. 1:136-138). The recombinant retrovirus alsocan be a hybrid virus such as that described in Choi, J K; Hoanga, N;Vilardi, A M; Conrad, P; Emerson, S G; Gewirtz, A M. (2001) HybridHIV/MSCV LTR Enhances Transgene Expression of Lentiviral Vectors inHuman CD34+ Hematopoietic Cells. Stem Cells 19, No. 3, 236-246.

[0064] In the preferred embodiment the transgene is incorporated into aviral construct that comprises an intact retroviral 5′ LTR and aself-inactivating 3′ LTR. The viral construct is preferably introducedinto a packaging cell line that packages viral genomic RNA based on theviral construct into viral particles with the desired host specificity.Viral particles are collected and used to infect the host cell. Each ofthese aspects is described in detail below.

[0065] The Viral Construct

[0066] The viral construct is a nucleotide sequence that comprisessequences necessary for the production of recombinant retrovirus in apackaging cell. In one embodiment the viral construct additionallycomprises genetic elements that allow for the desired expression of anRNA molecule or gene of interest in the host.

[0067] Generation of the viral construct can be accomplished using anysuitable genetic engineering techniques well known in the art,including, without limitation, the standard techniques of PCR,oligonucleotide synthesis, restriction endonuclease digestion, ligation,transformation, plasmid purification, and DNA sequencing, for example asdescribed in Sambrook et al. (Molecular Cloning: A Laboratory Manual.Cold Spring Harbor Laboratory Press, N.Y. (1989)), Coffin et al.(Retroviruses. Cold Spring Harbor Laboratory Press, N.Y. (1997)) and“RNA Viruses: A Practical Approach” (Alan J. Cann, Ed., OxfordUniversity Press, (2000)).

[0068] The viral construct may incorporate sequences from the genome ofany known organism. The sequences may be incorporated in their nativeform or may be modified in any way. For example, the sequences maycomprise insertions, deletions or substitutions. In the preferredembodiment the viral construct comprises sequences from a lentivirusgenome, such as the HIV genome or the SIV genome.

[0069] The viral construct preferably comprises sequences from the 5′and 3′ LTRs of a lentivirus. More preferably the viral constructcomprises the R and U5 sequences from the 5′ LTR of a lentivirus and aninactivated or self-inactivating 3′ LTR from a lentivirus. The LTRsequences may be LTR sequences from any lentivirus from any species. Forexample, they may be LTR sequences from HIV, SIV, FIV or BIV. Preferablythe LTR sequences are HIV LTR sequences. The virus also can incorporatesequences from MMV or MSCV.

[0070] The viral construct preferably comprises an inactivated orself-inactivating 3′ LTR. The 3′ LTR may be made self-inactivating byany method known in the art. In the preferred embodiment the U3 elementof the 3′ LTR contains a deletion of its enhancer sequence, preferablythe TATA box, Spl and NF-kappa B sites. As a result of theself-inactivating 3′ LTR, the provirus that is integrated into the hostcell genome will comprise an inactivated 5′ LTR.

[0071] Optionally, the U3 sequence from the lentiviral 5′ LTR may bereplaced with a promoter sequence in the viral construct. This mayincrease the titer of virus recovered from the packaging cell line. Anenhancer sequence may also be included. Any enhancer/promotercombination that increases expression of the viral RNA genome in thepackaging cell line may be used. In the preferred embodiment the CMVenhancer/promoter sequence is used (U.S. Pat. No. 5,168,062; Karasuyamaet al J. Exp. Med. 169:13 (1989).

[0072] The viral construct also comprises a transgene. The transgene,may be any nucleotide sequence, including sequences that serve asmarkers for the provirus. Preferably the transgene comprises one or moreRNA coding regions and/or one or more genes of interest. Schematicdiagrams of exemplary retroviral constructs are shown in FIGS. 1A and1B.

[0073] In the preferred embodiment the transgene comprises at least oneRNA coding region. Preferably the RNA coding region is a DNA sequencethat can serve as a template for the expression of a desired RNAmolecule in the host cell. In one embodiment, the viral constructcomprises two or more RNA coding regions.

[0074] The viral construct also preferably comprises at least one RNAPolymerase III promoter. The RNA Polymerase III promoter is operablylinked to the RNA coding region and can also be linked to a terminationsequence. In addition, more than one RNA Polymerase III promoter may beincorporated.

[0075] RNA Polymerase III promoters are well known to one of skill inthe art. A suitable range of RNA Polymerase III promoters can be found,for example, in Paule and White. Nucleic Acids Research., Vol 28, pp1283-1298 (2000), which is hereby incorporated by reference in itsentirety. The definition of RNA Polymerase III promoters also includeany synthetic or engineered DNA fragment that can direct RNA PolymeraseIII to transcribe a downstream RNA coding sequence. Further, the RNAPolymerase III (Pol III) promoter or promoters used as part of the viralvector can be inducible. Any suitable inducible Pol III promoter can beused with the methods of the invention. Particularly suited Pol IIIpromoters include the tetracycline responsive promoters provided inOhkawa and Taira Human Gene Therapy, Vol. 11, pp 577-585 (2000) and inMeissner et al. Nucleic Acids Research, Vol. 29, pp 1672-1682 (2001),which are incorporated herein by reference.

[0076] In one embodiment the transgene comprises a gene of interest thatencodes a protein that is desirably expressed in one or more cells of atransgenic animal, for example, a reporter or marker protein. Preferablythe gene of interest is located between the 5′ LTR and 3′ LTR sequences.Further, the gene of interest is preferably in a functional relationshipwith other genetic elements, for example transcription regulatorysequences such as promoters and/or enhancers, to regulate expression ofthe gene of interest in a particular manner once the transgene isincorporated into the host genome. In certain embodiments, the usefultranscriptional regulatory sequences are those that are highly regulatedwith respect to activity, both temporally and spatially.

[0077] Preferably the gene of interest is in a functional relationshipwith internal Polymerase II promoter/enhancer regulatory sequences. An“internal” promoter/enhancer is one that is located between the 5′ LTRand the 3′ LTR sequences in the viral construct and is operably linkedto the gene that is desirably expressed.

[0078] The Polymerase II promoter/enhancer may be any promoter, enhanceror promoter/enhancer combination known to increase expression of a genewith which it is in a functional relationship.

[0079] The internal promoter/enhancer is preferably selected based onthe desired expression pattern of the gene of interest and the specificproperties of known promoters/enhancers. Thus, the internal promoter maybe a constitutive promoter. Non-limiting examples of constitutivepromoters that may be used include the promoter for ubiquitin, CMV (U.S.Pat. No. 5,168,062; Karasuyama et al J. Exp. Med. 169:13 (1989), β-actin(Gunning et al. Proc. Natl. Acad. Sci. USA 84:4831-4835 (1987) and pgk(see, for example, U.S. Pat. Nos. 4,615,974 and 5,104,795; Adra et al.Gene 60:65-74 (1987), Singer-Sam et al. Gene 32:409-417 (1984) andDobson et al. Nucleic Acids Res. 10:2635-2637 (1982)). Alternatively,the promoter may be a tissue specific promoter. Several non-limitingexamples of tissue specific promoters that may be used include lck (see,for example, Garvin et al. Mol. Cell Biol. 8:3058-3064 (1988) andTakadera et al. Mol. Cell Biol. 9:2173-2180 (1989)), myogenin (Yee etal. Genes and Development 7:1277-1289 (1993), and thyl (Gundersen et al.Gene 113:207-214 (1992). In addition, promoters may be selected to allowfor inducible expression of the transgene. A number of systems forinducible expression using such a promoter are known in the art,including the tetracycline responsive system and the lacoperator-repressor system. It is also contemplated that a combination ofpromoters may be used to obtain the desired expression of the gene ofinterest. The skilled artisan will be able to select a promoter based onthe desired expression pattern of the gene in the resulting transgenicanimal.

[0080] An internal enhancer may also be present in the viral constructto increase expression of the gene of interest. For example the CMVenhancer (Karasuyama et al J. Exp. Med. 169:13 (1989) may be used incombination with the chicken β-actin promoter (see, e.g., JP1990005890-A1). Again, one of skill in the art will be able to selectthe appropriate enhancer based on the desired expression pattern.

[0081] The gene of interest is not limited in any way and includes anygene that the skilled practitioner desires to have integrated and/orexpressed in a transgenic animal. For example, the gene of interest maybe one that encodes a protein that serves as a marker to identify cellscomprising the provirus. In other embodiments the gene of interestencodes a protein that modifies a physical characteristic of thetransgenic animal, such as a protein that modifies size, growth, ortissue composition. In another example the gene of interest may encode aprotein of commercial value that may be harvested from the transgenicanimal.

[0082] In addition, more than one gene of interest may be placed infunctional relationship with the internal promoter. For example a geneencoding a marker protein may be placed after the primary gene ofinterest to allow for identification of cells that are expressing thedesired protein. In one embodiment a fluorescent marker protein,preferably green fluorescent protein (GFP), is incorporated into theconstruct along with the gene of interest. If a second reporter gene isincluded, an internal ribosomal entry site (IRES) sequence is alsopreferably included (U.S. Pat. No. 4,937,190). The IRES sequence mayfacilitate the expression of the reporter gene.

[0083] The viral construct may also contain additional genetic elements.The types of elements that may be included in the construct are notlimited in any way and will be chosen by the skilled practitioner toachieve a particular result. For example, a signal that facilitatesnuclear entry of the viral genome in the target cell may be included. Anexample of such a signal is the HIV-1 flap signal.

[0084] Further, elements may be included that facilitate thecharacterization of the provirus integration site in the genome of theanimal. For example, a tRNA amber suppressor sequence may be included inthe construct.

[0085] In addition, the construct may contain one or more geneticelements designed to enhance expression of the gene of interest. Forexample, a woodchuck hepatitis virus responsive element (WRE) may beplaced into the construct (Zufferey et al. J. Virol. 74:3668-3681(1999); Deglon et al. Hum. Gene Ther.11:179-190 (2000)).

[0086] A chicken β-globin insulator (Chung et al. Proc. Natl. Acad. Sci.USA 94:575-580 (1997)) may also be included in the viral construct. Thiselement has been shown to reduce the chance of silencing the integratedprovirus in the transgenic animal due to methylation andheterochromatinization effects. In addition, the insulator may shieldthe internal enhancer, promoter and exogenous gene from positive ornegative positional effects from surrounding DNA at the integration siteon the chromosome.

[0087] Any additional genetic elements are preferably inserted 3′ of thegene of interest.

[0088] In a specific embodiment, the viral vector comprises: acytomegalovirus (CMV) enhancer/promoter sequence; the R and U5 sequencesfrom the HIV 5′ LTR; the HIV-1 flap signal; an internal enhancer; aninternal promoter; a gene of interest; the woodchuck hepatitis virusresponsive element; a tRNA amber suppressor sequence; a U3 element witha deletion of its enhancer sequence; the chicken β-globin insulator; andthe R and U5 sequences of the 3′ HIV LTR.

[0089] The viral construct is preferably cloned into a plasmid that maybe transfected into a packaging cell line. The preferred plasmidpreferably comprises sequences useful for replication of the plasmid inbacteria.

[0090] Production of Virus

[0091] Any method known in the art may be used to produce infectiousretroviral particles whose genome comprises an RNA copy of the viralconstruct described above.

[0092] Preferably, the viral construct is introduced into a packagingcell line. The packaging cell line provides the viral proteins that arerequired in trans for the packaging of the viral genomic RNA into viralparticles. The packaging cell line may be any cell line that is capableof expressing retroviral proteins. Preferred packaging cell linesinclude 293 (ATCC CCL X), HeLa (ATCC CCL 2), D17 (ATCC CCL 183), MDCK(ATCC CCL 34), BHK (ATCC CCL-10) and Cf2Th (ATCC CRL 1430). The mostpreferable cell line is the 293 cell line.

[0093] The packaging cell line may stably express the necessary viralproteins. Such a packaging cell line is described, for example, in U.S.Pat. No. 6,218,181. Alternatively a packaging cell line may betransiently transfected with plasmids comprising nucleic acid thatencodes the necessary viral proteins.

[0094] In one embodiment a packaging cell line that stably expresses theviral proteins required for packaging the RNA genome is transfected witha plasmid comprising the viral construct described above.

[0095] In another embodiment a packaging cell line that does not stablyexpress the necessary viral proteins is co-transfected with two or moreplasmids essentially as described in Yee et al. (Methods Cell. Biol.43A, 99-112 (1994)). One of the plasmids comprises the viral constructcomprising the transgene. The other plasmid(s) comprises nucleic acidencoding the proteins necessary to allow the cells to produce functionalvirus that is able to infect the desired host cell.

[0096] The packaging cell line may not express envelope gene products.In this case the packaging cell line will package the viral genome intoparticles that lack an envelope protein. As the envelope protein isresponsible, in part, for the host range of the viral particles, theviruses are preferably pseudotyped. Thus the packaging cell line ispreferably transfected with a plasmid comprising sequences encoding amembrane-associated protein that will permit entry of the virus into ahost cell. One of skill in the art will be able to choose theappropriate pseudotype for the host cell that is to be used. Forexample, in one embodiment the viruses are pseudotyped with thevesicular stomatitis virus envelope glycoprotein (VSVg). In addition toconferring a specific host range this pseudotype may permit the virus tobe concentrated to a very high titer. Viruses can alternatively bepseudotyped with ecotropic envelope proteins that limit infection to aspecific species, such as mice or birds. For example, in anotherembodiment, a mutant ecotropic envelope protein is used, such as theecotropic envelope protein 4.17 (Powell et al. Nature Biotechnology18(12):1279-1282 (2000)).

[0097] In the preferred embodiment a packaging cell line that does notstably express viral proteins is transfected with the viral construct, asecond vector comprising the HIV-1 packaging vector with the env, nef,5′LTR, 3′LTR and vpu sequences deleted, and a third vector encoding anenvelope glycoprotein. Preferably the third vector encodes the VSVgenvelope glycoprotein.

[0098] In another embodiment of invention, RNA interference activity ofthe packaging cells is suppressed to improve the production ofrecombinant virus. This includes, without limitation, the use ofcotransfection or stable transfection of constructs expressing siRNAmolecules to inhibit Dicer, an RNase III family member of ribonucleasewhich is essential for RNA interference (Hammond et al. Nat. Rev. Genet.2:110-119 (2001)).

[0099] The recombinant virus is then preferably purified from thepackaging cells, titered and diluted to the desired concentration.

[0100] Transgenic Animals

[0101] In order to make transgenic animals, an oocyte or one or moreembryonic cells are infected with the recombinant virus produced asdescribed above. One of skill in the art will recognize that the methodof infection and the treatment of the cell following infection willdepend upon the type of animal from which the cell is obtained. Forexample, mammalian cells are preferably implanted in a pseudopregnantfemale following infection while for the generation of transgenic birdsor fish, the virus is preferably delivered to a laid egg and thusimplantation is not required.

[0102] While early methods of making transgenic animals required thecells to be rapidly dividing, there is no such requirement in themethods of the present invention. Thus the cell may be contacted at anypoint in development. In the preferred embodiment, a zygote is contactedwith the recombinant virus.

[0103] The cells to be infected with the virus may be obtained by anymethod known in the art and appropriate for the specific species inwhich it is desired to make a transgenic animal. For example, therecovery of fertilized mouse oocytes is described in Hogan et al.(Manipulating the Mouse Embryo: A Laboratory Manual. 2^(nd) ed. ColdSpring Harbor Laboratory Press, NY (1994)). A method for obtainingfertilized rat oocytes is described in Armstrong et al. (Biol. Reprod.39,511-518 (1998)).

[0104] It is not necessary that the cells be contacted afterfertilization. In one embodiment, the virus is delivered to unfertilizedova. Development may then be initialized, for example by in vitrofertilization.

[0105] Delivery of the Virus

[0106] The virus may be delivered to the cell in any way that allows thevirus to infect the cell. Preferably the virus is allowed to contact thecell membrane. Two preferred methods of delivering the virus tomammalian cells, injection and direct contact, are described below.

[0107] Injection

[0108] In a first embodiment the virus is injected into theperivitelline space between the zona pellucida and the cell membrane ofa single-cell zygote. Preferably less than 50 picoliters of viralsuspension is injected, more preferably less than 25 picoliters and evenmore preferably about 10 picoliters.

[0109] The virus is preferably present in a viral suspension and may beinjected by any method known in the art. The viral suspension ispreferably injected through a hydraulic injector. More preferably aglass micropipette is used to inject the virus. In one embodiment amicropipette is prepared by pulling borosilicate glass capillary on apipette puller. The tip is preferably opened and beveled toapproximately 10 μm. The lentiviral suspension may be loaded into themicropipette from the tip using gentle negative pressure.

[0110] In one embodiment the cell is stabilized with a holding pipettemounted on a micromanipulator, such as by gentle negative pressureagainst a fire-polished pipette, and a second micromanipulator is usedto direct the tip of a micropipette into the space between the zonapellucida and the cell membrane, where the virus is injected.

[0111] Direct Contact

[0112] In another embodiment the zona pellucida is removed from the cellto produce a denuded embryo and the cell membrane is contacted with thevirus. The zona pellucida may be removed by any method known in the art.Preferably it is removed by enzymatic treatment. For example, treatmentwith pronase may be used to remove the zona pellucida while the cellmembrane is kept intact. Alternatively, the cell may be placed in mediaat pH at which the zona pellucida dissolves while the cell membraneremains intact. For example the cell may be incubated in an acidicTyrode's solution at room temperature for several minutes. Once the zonapellucida is removed, any method that allows for the virus to contactthe cell membrane may be used. Preferably, the cell is incubated in asolution containing the virus. Even more preferably, the solution ismedia that facilitates survival of the cell.

[0113] In this embodiment, the cells are preferably contacted with thevirus in culture plates. The virus may be suspended in media and addedto the wells of a multi-well culture plate. The cells may then be platedin the individual wells. The media containing the virus may be addedprior to the plating of the cells or after the cells have been plated.Preferably individual cells are incubated in approximately 10 μl ofmedia. However, any amount of media may be used as long as anappropriate concentration of virus in the media is maintained such thatinfection of the host cell occurs.

[0114] The cells are preferably incubated with the virus for asufficient amount of time to allow the virus to infect the cells.Preferably the cells are incubated with virus for at least 1 hour, morepreferably at least 5 hours and even more preferably at least 10 hours.

[0115] Both the injection and direct contact embodiments mayadvantageously be scaled up to allow high throughput transgenesis.Because of the relative simplicity of the injection technique, it ispossible to inject many embryos rapidly. For example, it is possible toinject more than 200 fertilized oocytes in less than one hour. Withregard to the direct contact embodiment, any number of embryos may beincubated in the viral suspension simultaneously. This may beaccomplished, for example, by planting the desired number of single-cellzygotes in multi-well tissue culture plates containing the virussuspended in media appropriate for the survival and growth of the cells.

[0116] In both embodiments, any concentration of virus that issufficient to infect the cell may be used. Preferably the concentrationis at least 1 pfu/μl, more preferably at least 10 pfu/μl, even morepreferably at least 400 pfu/μl and even more preferably at least 1×10⁴pfu/μl.

[0117] Following infection with the virus, the cells are preferablyimplanted in an animal. More preferably cells infected with the virusare implanted in pseudo-pregnant animals of the same species from whichthe infected cells were obtained. Methods of creating pseudo-pregnancyin animals and implanting embryos are well known in the art and aredescribed, for example, in Hogan et al. (Manipulating the Mouse Embryo:A Laboratory Manual. 2^(nd) ed. Cold Spring Harbor Laboratory Press, NY(1994)).

[0118] In the preferred embodiment early stage embryos (approximately0-2.5 days p.c.) still with an intact zona pellucida are transferred tothe oviduct of timed pseudopregnant female (preferably 0.5 days p.c.),while embryos that have reached the blastocyst stage are transferred tothe uterus of timed pseudopregnant females (preferably 2.5 days p.c.).Denuded embryos are preferably cultured in vitro until they reach themorula or blastocyst stage (48 to 72 hours in culture), and are thenimplanted into appropriately timed pseudopregnant females.

[0119] The embryos and resulting animals may be analyzed, for examplefor integration of the transgene, the number of copies of the transgenethat integrated, the location of the integration, the ability totransmit the transgene to progeny and expression of the transgene. Suchanalysis may be carried out at any time and may be carried out by anymethods known in the art. Standard techniques are described, forexample, in Hogan et al. (supra).

[0120] The methods of infecting cells disclosed above do not depend uponspecies-specific characteristics of the cells. As a result, they arereadily extended to all mammalian species.

[0121] Initial experiments with mice indicate that of those animals thatdevelop to full term, 80-90% carried at least one copy of the transgeneand that, of these, approximately 85% express the gene of interest. Ofthe transgenic animals about 25% carry only 1 or 2 copies of thetransgene. The highest number of proviral insertions observed was about30. Of the animals that carried only 1 or 2 copies of the transgene,about 80% expressed the gene of interest.

[0122] As discussed above, the modified retrovirus can be pseudotyped toconfer upon it a broad host range. One of skill in the art would also beaware of appropriate internal promoters to achieve the desiredexpression of a gene of interest in a particular animal species. Thus,one of skill in the art will be able to modify the method of infectingcells to create transgenic animals of any species.

[0123] In one embodiment, transgenic birds are created by delivering amodified retrovirus, as described above, to the primordial germ cells ofearly stage avian embryos. Freshly laid eggs are obtained and placed ina temperature controlled, humidified incubator. Preferably, theembryonic blastodisc in the egg is gradually rotated to lie on top ofthe yolk. This may be accomplished by any method known in the art, suchas by gently rocking the egg regularly, preferably every 15 minutes.Approximately 36 hours later, the modified retrovirus is delivered intothe space between the embryonic disk and the perivitelline membrane.Preferably about 50 nL of viral solution is delivered, more preferablyabout 100 nL of viral solution is delivered, and even more preferablyabout 200 nL of viral solution is delivered. The viral solution may bedelivered by any method known in the art for delivering compositions tothe inside of an egg. In the preferred embodiment a window is opened inthe shell, the viral solution is injected through the window and theshell window is closed. The eggs are preferably incubated untilhatching. The eggs will hatch after approximately 20 days, dependingupon the particular avian species from which they are obtained. Hatchedchicks are preferably raised to sexual maturity and mated. Thetransgenic offspring of the founder animals may be identified by anymethod known in the art, such as Southern blot, PCR and expressionanalysis.

[0124] In another embodiment, transgenic fish are created by deliveringthe modified retrovirus, described above, to single-cell fish embryos.Fertilized fish eggs are collected by any method known in the art. Themodified retrovirus is then preferably delivered to the space betweenthe chorion and the cell membrane. This may be accomplished, forexample, by loading the modified retrovirus in solution into a glasspipette. The pipette may then be used to pierce the chorion membrane anddeliver the viral suspension. Preferably about 50 nL of viral solutionis delivered, more preferably about 100 nL of viral solution isdelivered, and even more preferably about 200 nL of viral solution isdelivered. Injected embryos are preferably returned to atemperature-controlled water tank and allowed to mature. At sexualmaturity the founder fish are preferably mated and their progenyanalyzed for the presence of the transgene by any method known in theart.

[0125] As mentioned above, the methods of the present invention willalso prove useful in techniques for identifying genes that are involvedin specific biological processes, such as gene trap assays andlarge-scale mutagenesis screens. Such methods are described in thecopending provisional patent applications 60/322,031 filed on Sep. 13,2001 and copending U.S. provisional patent application No. 60/347,782filed on Jan. 9, 2002.

[0126] Down-Regulating Gene Expression in a Target Cell

[0127] The methods described herein allow the expression of RNAmolecules in cells, and are particularly suited to the expression ofsmall RNA molecules, which can not be readily expressed from a Pol IIpromoter. According to a preferred embodiment of the invention, an RNAmolecule is expressed within a cell in order to down-regulate theexpression of a target gene. The ability to down-regulate a target genehas many therapeutic and research applications, including identifyingthe biological functions of particular genes. Using the techniques andcompositions of the invention, it will be possible to knock-down (ordown-regulate) the expression of a large number of genes, both in cellculture and in mammalian organisms.

[0128] In preferred embodiments of the invention, an RNA expressioncassette comprises a Pol III promoter and an RNA coding region. The RNAcoding region preferably encodes an RNA molecule that is capable ofdown-regulating the expression of a particular gene or genes. The RNAmolecule encoded can, for example, be complementary to the sequence ofan RNA molecule encoding a gene to be down-regulated. In such anembodiment, the RNA molecule preferably acts through an antisensemechanism.

[0129] A more preferred embodiment involves the expression of adouble-stranded RNA complex, or an RNA molecule having a stem-loop or aso-called “hairpin” structure. As used herein, the term “RNA duplex”refers to the double stranded regions of both the RNA complex and thedouble-stranded region of the hairpin or stem-lop structure.

[0130] Double stranded RNA has been shown to inhibit gene expression ofgenes having a complementary sequence through a process termed RNAinterference or suppression (see, for example, Hammond et al. Nat. Rev.Genet. 2:110-119 (2001)).

[0131] According to the invention, an RNA duplex or siRNA correspondingto a region of a gene to be down-regulated is expressed in the cell. TheRNA duplex is substantially identical (typically at least about 80%identical, more preferably at least about 90% identical) in sequence tothe sequence of the gene targeted for down regulation. siRNA duplexesare described, for example, in Bummelkamp et al. Science 296:550-553(2202), Caplen et al. Proc. Natl. Acad. Sci. USA 98:9742-9747 (2001) andPaddison et al. Genes & Devel. 16:948-958 (2002).

[0132] The RNA duplex is generally at least about 15 nucleotides inlength and is preferably about 15 to about 30 nucleotides in length.However, a significantly longer RNA duplex can be used effectively insome organisms. In a more preferred embodiment, the RNA duplex isbetween about 19 and 22 nucleotides in length. The RNA duplex ispreferably identical to the target nucleotide sequence over this region.

[0133] When the gene to be down regulated is in a family of highlyconserved genes, the sequence of the duplex region can be chosen withthe aid of sequence comparison to target only the desired gene. On theother hand, if there is sufficient identity among a family of homologousgenes within an organism, a duplex region can be designed that woulddown regulate a plurality of genes simultaneously.

[0134] The duplex RNA can be expressed in a cell from a singleretroviral construct. In the preferred embodiment, a single RNA codingregion in the construct is a serves as a template for the expression ofa self-complementary hairpin RNA, comprising a sense region, a loopregion and an antisense region. This embodiment is illustrated in FIG.2, which shows a schematic view of an RNA expression cassette having anRNA Pol III promoter 100 operatively linked to an RNA coding region,having a sense region 110, a loop region 120, an antisense region 130and a terminator region 140. The sense 110 and antisense 130 regions areeach preferably about 15 to about 30 nucleotides in length. The loopregion 120 preferably is about 2 to about 15 nucleotides in length, morepreferably from about 4 to about 9 nucleotides in length. Followingexpression the sense and antisense regions form a duplex.

[0135] In another embodiment, the retroviral construct comprises two RNAcoding regions. The first coding region is a template for the expressionof a first RNA and the second coding region is a template for theexpression of a second RNA. Following expression, the first and secondRNA's form a duplex. The retroviral construct preferably also comprisesa first Pol III promoter operably linked to the first RNA coding regionand a second Pol III promoter operably linked to the second RNA codingregion. This embodiment is illustrated in FIG. 3, which shows aschematic view of an RNA expression cassette having an RNA PolymeraseIII promoter 100 linked to a first RNA coding region 110 and a firstterminator sequence 140 and a second RNA polymerase III promoter 105linked to a second RNA coding region 115 and a second terminator 145.

[0136] In yet another embodiment of the invention, the retroviralconstruct comprises a first RNA Pol III promoter operably linked to afirst RNA coding region, and a second RNA Pol III promoter operablylinked to the same first RNA coding region in the opposite direction,such that expression of the RNA coding region from the first RNA Pol IIIpromoter results in a synthesis of a first RNA molecule as the sensestrand and expression of the RNA coding region from the second RNA PolIII promoter results in synthesis of a second RNA molecule as anantisense strand that is substantially complementary to the first RNAmolecule. In one such embodiment, both RNA Polymerase III promoters areseparated from the RNA coding region by termination sequences,preferably termination sequences having five consecutive T residues.FIG. 4 shows a schematic view of an RNA expression cassette having afirst RNA Polymerase III promoter 100 linked to an RNA coding region 110and a first terminator sequence 145. The expression cassette has asecond RNA polymerase III promoter 105 linked to the RNA coding region115, the same sequence as 110 in reverse, and a second terminator 140.

[0137] In further embodiments an RNA duplex is expressed using two ormore retroviral constructs. In one embodiment, a first retroviralconstruct is used that directs the expression of a first RNA and asecond retroviral construct is used that directs expression of a secondRNA that is complementary to the first. Following expression the firstand second RNAs form a duplex region. It is preferred, however, that theentire duplex region is introduced using retroviral particles derivedfrom a single retroviral construct. As discussed above, severalstrategies for expressing a duplex RNA from a single viral construct areshown in FIGS. 2-4.

[0138] The RNA duplexes may be flanked by single stranded regions on oneor both sides of the duplex. For example, in the case of the hairpin,the single stranded loop region would connect the duplex region at oneend.

[0139] The RNA coding region is generally operatively linked to aterminator sequence. The pol III terminators preferably comprise ofstretches of 4 or more thymidine (“T”) residues. In a preferredembodiment, a cluster of 5 consecutive Ts is linked immediatelydownstream of the RNA coding region to serve as the terminator. In sucha construct pol III transcription is terminated at the second or third Tof the DNA template, and thus only 2 to 3 uridine (“U”) residues areadded to the 3′ end of the coding sequence.

[0140] The sequence of the RNA coding region, and thus the sequence ofthe RNA duplex, preferably is chosen to be complementary to the sequenceof a gene whose expression is to be downregulated in a cell or organism.The degree of down regulation achieved with a given RNA duplex sequencefor a given target gene will vary by sequence. One of skill in the artwill be able to readily identify an effective sequence. For example, inorder to maximize the amount of suppression in a transgenic animal, anumber of sequences can be tested for their efficacy in cell cultureprior to generating a transgenic animal.

[0141] The methods of the present invention will find great commercialapplication, for example in biotechnology, medicine and agriculture. Forexample, in agriculture the described methods may be used to conferdisease resistance by expressing in a cell or organism an siRNA thatspecifically down-regulates the expression of a gene associated with apathogen or disease state. In biotechnology, the ability to rapidlydevelop large numbers of transgenic animals with desired modulation ofspecific genes will allow for the analysis of gene function and theevaluation of compounds that potentially modulate gene expression,protein function, and are useful in treating a disease or disorder. Inparticular, by observing the effect of down-regulating specific genes intransgenic animals, the biological function of those genes may bedetermined. In medicine the methods of the invention may be used totreat patients suffering from particular diseases or disorders, such asHIV, or to confer immunity or resistance to particular pathogens. Forexample, specific cells may be infected in vivo or ex vivo withrecombinant retrovirus encoding an siRNA that down-regulates theactivity of a gene whose activity is associated with a particulardisease or disorder.

[0142] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way. Indeed, various modifications of the invention in additionto those shown and described herein will become apparent to thoseskilled in the art from the foregoing description and fall within thescope of the appended claims.

[0143] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES Example 1

[0144] An lentiviral construct was constructed by insertion of an siRNAexpression cassette into the PacI site of HC-FUGW vector (FIG. 5; SEQ IDNO: 2). The siRNA was designed to down-regulate expression of the lacZgene. The HC-FUGW vector comprised a GFP marker gene operably linked tothe human Ubiquitin promoter. The GFP marker was useful for trackingtransduction events. The vector also comprised an HIV DNA Flap elementto improve the virus titers, and the WRE for high level expression ofviral genes. The siRNA expression cassette was composed of a pol IIIpromoter and a small hairpin RNA coding region followed by a pol IIIterminator site. The pol III promoter (SEQ ID NO:3) was derived from the−240 to −9 region of human HI-RNA promoter and was cloned as an Eco RIfragment by PCR amplification from HEK293 genomic DNA. The pol IIIpromoter was connected to the downstream RNA coding region by a 7 basepair linker sequence to ensure that the transcription was preciselyinitiated at the first nucleotide of the RNA coding sequence. The smallhairpin RNA coding region comprised a 19 nt sequence corresponding tothe 1900-1918 region of the sense strand of the bacterialbeta-galactosidase (lacZ) gene coding sequence and the 19 nt perfectreverse complementary sequence separated by a 9 nt loop region. Theterminator was comprised of 5 consecutive thymidine residues linkedimmediately downstream of the RNA coding sequence. The sequence of thehairpin siRNA is shown in SEQ ID NO: 1.

Example 2

[0145] Transduction of cultured mammalian cells with retrovirus derivedfrom the retroviral construct described in Example 1 was achieved (FIG.6). The retroviral vector encoding a small hairpin RNA moleculedescribed in Example 1, was used to transfect cultured mammalian cellsthat express lacZ. A profound decrease in the expression of the lacZ wasobserved.

[0146] The lacZ siRNA virus was produced by cotransfection of theretroviral vector, a helper virus plasmid and VSVg expression plasmid inHEK293 cells. The virus particles were harvested from the cell culturesupernatants and concentrated by ultracentrifugation. The concentratedvirus preparations were used to infect either mouse embryonicfibroblasts (MEF) or HEK293 cells which harbor both lacZ and fireflyluciferase (Luc) reporter genes. Infection was monitored by the GFPsignal which is expressed from the marker gene cassette of the viralvector. Under the conditions of this experiment, >98% of the test cellswere GPF+ and thus were successfully transduced. The expression levelsof lacZ and Luc reporter genes were measured by chemiluminescent assaysusing commercially available kits (lacZ assay kit from Roche and Lucfrom Promega). The lacZ siRNA virus only inhibited the expression oflacZ but not Luc. The specific inhibition was determined by the ratio oflacZ activity to Luc activity. The lacZ/Luc ratio of the uninfectedparental cells was arbitrarily set to 1 and the values of the infectedcells were calculated accordingly. As shown in FIG. 6, transfection withthe virus resulted in dramatic reduction in the amount of expression ofthe lacZ gene in both MEK and HEK293 cells.

[0147] A tet-iducible lacZ siRNA lentiviral vector was also prepared asillustrated in FIG. 8. A Tet repressor gene (TetR; SEQ ID NO: 5) wasplaced the under the control of the human UbiquitinC promoter so thatits expression could be monitored by the downstream GFP marker. Theanti-lacZ siRNA cassette was driven by a Tet-inducible pol III promoterderived from human U6-promoter (−328 to +1) containing a single TetRbinding site (TetO1) between the PSE and TATA box (SEQ ID NO: 4). TheTetR coding sequence was PCR amplified from genomic DNA from the TOP10strain of E. coli adn cloned into a modified version of FUIGW as aBgl2-EcoR1 fragment. In the absence of tetracycline, TetR binds to thepromoter and its expression is repressed. Upon the addition oftetracycline, TetR is moved from the promoter and transcription starts.

[0148] The Tet-inducible siRNA expression cassette was able to regulategene expression in response to Doxycycline treatment. Virus was preparedfrom the retroviral construct carrying the Tet-inducible lacZ-siRNAcassette and a Tet repressor under the control of a UbiquitinC promoterand used to transduce HEK293 cells expressing both lacZ and luciferase(293Z+Luc). The transduced cells were treated with 10 ug/ml Doxycycline(Plus Dox) for 48 hr or without the Doxycycline treatment as a control(No Dox). LacZ and luciferase activities were measured as described inthe previous figures. The relative suppression activity is calculated asthe ratio of lacZ versus luciferase and No Dox control was arbitrarilyset to 1. As can be seen in FIG. 9, in the presence of doxycyclinesuppression of lacZ activity was significantly enhanced.

Example 3

[0149] This example demonstrates the generation of transgenic animalsthat express an siRNA molecule encoded by a lentiviral vector. Theexpression of the lacZ specific siRNA described in Example 1 resulted inextensive suppression of lacZ activity in ROSA26+/− mice.

[0150] ROSA26+/− mice carry one copy of a ubiquitously expressed lacZreporter gene. The lacZ siRNA virus preparations described in Example 2were used for perivitelline injection of ROSA26+/− single cell embryosobtained from hormone primed C57B1/6 female donors x ROSA26+/+ studmales. The injected single cell embryos were subsequently transferredinto the oviduct of timed pseudopregnant female recipients. Embryonicday 15.5 to 17.5 (E15.5-17.5) fetuses were recovered from the surrogatemothers. Successful transgenesis was scored by positive GFP signalobserved with the fetuses under fluorescent microscope. Protein extractsprepared from the limb tissues of the fetuses were used for the LacZchemiluminescent assay according to the manufacturer's instruction(Roche), and protein concentrations of the tissue extracts weredetermined by the Bradford assay (BioRad). The lacZ expression levelswere expressed as light units (LU) per ug of proteins (LU/ug). TheE15.5-17.5 fetuses from the timed mating of C57B1/6 females×ROSA26+/+males and C57B1/6 females×C57B1/6 males were served as the positive andnegative controls respectively. The results are shown in FIG. 7. AnimalsG1-G4 (those treated derived from embroys infected with the viruscomprising the siRNA construct) showed markedly decreased expression ofthe lacZ gene as compared with untreated control animals.

1 5 1 59 DNA Artificial Sequence This represents a siRNA cassettecomprising bacterial sequence and synthetic linker, loop and terminatorsequences. 1 gatccccgtg accagcgaat acctgtttca agagaacagg tattcgctggtcacttttt 59 2 9941 DNA Artificial Sequence This sequence represents alentiviral vector comprising a human immunodeficiency virus flapsequence, a green fluorescent protein variant sequence, a humanubiquitin promoter sequence and a woodchuck hepatitis regulator elementsequence. 2 gtcgacggat cgggagatct cccgatcccc tatggtgcac tctcagtacaatctgctctg 60 atgccgcata gttaagccag tatctgctcc ctgcttgtgt gttggaggtcgctgagtagt 120 gcgcgagcaa aatttaagct acaacaaggc aaggcttgac cgacaattgcatgaagaatc 180 tgcttagggt taggcgtttt gcgctgcttc gcgatgtacg ggccagatatacgcgttgac 240 attgattatt gactagttat taatagtaat caattacggg gtcattagttcatagcccat 300 atatggagtt ccgcgttaca taacttacgg taaatggccc gcctggctgaccgcccaacg 360 acccccgccc attgacgtca ataatgacgt atgttcccat agtaacgccaatagggactt 420 tccattgacg tcaatgggtg gagtatttac ggtaaactgc ccacttggcagtacatcaag 480 tgtatcatat gccaagtacg ccccctattg acgtcaatga cggtaaatggcccgcctggc 540 attatgccca gtacatgacc ttatgggact ttcctacttg gcagtacatctacgtattag 600 tcatcgctat taccatggtg atgcggtttt ggcagtacat caatgggcgtggatagcggt 660 ttgactcacg gggatttcca agtctccacc ccattgacgt caatgggagtttgttttggc 720 accaaaatca acgggacttt ccaaaatgtc gtaacaactc cgccccattgacgcaaatgg 780 gcggtaggcg tgtacggtgg gaggtctata taagcagcgc gttttgcctgtactgggtct 840 ctctggttag accagatctg agcctgggag ctctctggct aactagggaacccactgctt 900 aagcctcaat aaagcttgcc ttgagtgctt caagtagtgt gtgcccgtctgttgtgtgac 960 tctggtaact agagatccct cagacccttt tagtcagtgt ggaaaatctctagcagtggc 1020 gcccgaacag ggacttgaaa gcgaaaggga aaccagagga gctctctcgacgcaggactc 1080 ggcttgctga agcgcgcacg gcaagaggcg aggggcggcg actggtgagtacgccaaaaa 1140 ttttgactag cggaggctag aaggagagag atgggtgcga gagcgtcagtattaagcggg 1200 ggagaattag atcgcgatgg gaaaaaattc ggttaaggcc agggggaaagaaaaaatata 1260 aattaaaaca tatagtatgg gcaagcaggg agctagaacg attcgcagttaatcctggcc 1320 tgttagaaac atcagaaggc tgtagacaaa tactgggaca gctacaaccatcccttcaga 1380 caggatcaga agaacttaga tcattatata atacagtagc aaccctctattgtgtgcatc 1440 aaaggataga gataaaagac accaaggaag ctttagacaa gatagaggaagagcaaaaca 1500 aaagtaagac caccgcacag caagcggccg ctgatcttca gacctggaggaggagatatg 1560 agggacaatt ggagaagtga attatataaa tataaagtag taaaaattgaaccattagga 1620 gtagcaccca ccaaggcaaa gagaagagtg gtgcagagag aaaaaagagcagtgggaata 1680 ggagctttgt tccttgggtt cttgggagca gcaggaagca ctatgggcgcagcgtcaatg 1740 acgctgacgg tacaggccag acaattattg tctggtatag tgcagcagcagaacaatttg 1800 ctgagggcta ttgaggcgca acagcatctg ttgcaactca cagtctggggcatcaagcag 1860 ctccaggcaa gaatcctggc tgtggaaaga tacctaaagg atcaacagctcctggggatt 1920 tggggttgct ctggaaaact catttgcacc actgctgtgc cttggaatgctagttggagt 1980 aataaatctc tggaacagat ttggaatcac acgacctgga tggagtgggacagagaaatt 2040 aacaattaca caagcttaat acactcctta attgaagaat cgcaaaaccagcaagaaaag 2100 aatgaacaag aattattgga attagataaa tgggcaagtt tgtggaattggtttaacata 2160 acaaattggc tgtggtatat aaaattattc ataatgatag taggaggcttggtaggttta 2220 agaatagttt ttgctgtact ttctatagtg aatagagtta ggcagggatattcaccatta 2280 tcgtttcaga cccacctccc aaccccgagg ggacccgaca ggcccgaaggaatagaagaa 2340 gaaggtggag agagagacag agacagatcc attcgattag tgaacggatcggcactgcgt 2400 gcgccaattc tgcagacaaa tggcagtatt catccacaat tttaaaagaaaaggggggat 2460 tggggggtac agtgcagggg aaagaatagt agacataata gcaacagacatacaaactaa 2520 agaattacaa aaacaaatta caaaaattca aaattttcgg gtttattacagggacagcag 2580 agatccagtt tggttaatta agggtgcagc ggcctccgcg ccgggttttggcgcctcccg 2640 cgggcgcccc cctcctcacg gcgagcgctg ccacgtcaga cgaagggcgcaggagcgttc 2700 ctgatccttc cgcccggacg ctcaggacag cggcccgctg ctcataagactcggccttag 2760 aaccccagta tcagcagaag gacattttag gacgggactt gggtgactctagggcactgg 2820 ttttctttcc agagagcgga acaggcgagg aaaagtagtc ccttctcggcgattctgcgg 2880 agggatctcc gtggggcggt gaacgccgat gattatataa ggacgcgccgggtgtggcac 2940 agctagttcc gtcgcagccg ggatttgggt cgcggttctt gtttgtggatcgctgtgatc 3000 gtcacttggt gagttgcggg ctgctgggct ggccggggct ttcgtggccgccgggccgct 3060 cggtgggacg gaagcgtgtg gagagaccgc caagggctgt agtctgggtccgcgagcaag 3120 gttgccctga actgggggtt ggggggagcg cacaaaatgg cggctgttcccgagtcttga 3180 atggaagacg cttgtaaggc gggctgtgag gtcgttgaaa caaggtggggggcatggtgg 3240 gcggcaagaa cccaaggtct tgaggccttc gctaatgcgg gaaagctcttattcgggtga 3300 gatgggctgg ggcaccatct ggggaccctg acgtgaagtt tgtcactgactggagaactc 3360 gggtttgtcg tctggttgcg ggggcggcag ttatgcggtg ccgttgggcagtgcacccgt 3420 acctttggga gcgcgcgcct cgtcgtgtcg tgacgtcacc cgttctgttggcttataatg 3480 cagggtgggg ccacctgccg gtaggtgtgc ggtaggcttt tctccgtcgcaggacgcagg 3540 gttcgggcct agggtaggct ctcctgaatc gacaggcgcc ggacctctggtgaggggagg 3600 gataagtgag gcgtcagttt ctttggtcgg ttttatgtac ctatcttcttaagtagctga 3660 agctccggtt ttgaactatg cgctcggggt tggcgagtgt gttttgtgaagttttttagg 3720 caccttttga aatgtaatca tttgggtcaa tatgtaattt tcagtgttagactagtaaag 3780 cttctgcagg tcgactctag aaaattgtcc gctaaattct ggccgtttttggcttttttg 3840 ttagacagga tccccgggta ccggtcgcca ccatggtgag caagggcgaggagctgttca 3900 ccggggtggt gcccatcctg gtcgagctgg acggcgacgt aaacggccacaagttcagcg 3960 tgtccggcga gggcgagggc gatgccacct acggcaagct gaccctgaagttcatctgca 4020 ccaccggcaa gctgcccgtg ccctggccca ccctcgtgac caccctgacctacggcgtgc 4080 agtgcttcag ccgctacccc gaccacatga agcagcacga cttcttcaagtccgccatgc 4140 ccgaaggcta cgtccaggag cgcaccatct tcttcaagga cgacggcaactacaagaccc 4200 gcgccgaggt gaagttcgag ggcgacaccc tggtgaaccg catcgagctgaagggcatcg 4260 acttcaagga ggacggcaac atcctggggc acaagctgga gtacaactacaacagccaca 4320 acgtctatat catggccgac aagcagaaga acggcatcaa ggtgaacttcaagatccgcc 4380 acaacatcga ggacggcagc gtgcagctcg ccgaccacta ccagcagaacacccccatcg 4440 gcgacggccc cgtgctgctg cccgacaacc actacctgag cacccagtccgccctgagca 4500 aagaccccaa cgagaagcgc gatcacatgg tcctgctgga gttcgtgaccgccgccggga 4560 tcactctcgg catggacgag ctgtacaagt aaagcggccg cgactctagaattcgatatc 4620 aagcttatcg ataatcaacc tctggattac aaaatttgtg aaagattgactggtattctt 4680 aactatgttg ctccttttac gctatgtgga tacgctgctt taatgcctttgtatcatgct 4740 attgcttccc gtatggcttt cattttctcc tccttgtata aatcctggttgctgtctctt 4800 tatgaggagt tgtggcccgt tgtcaggcaa cgtggcgtgg tgtgcactgtgtttgctgac 4860 gcaaccccca ctggttgggg cattgccacc acctgtcagc tcctttccgggactttcgct 4920 ttccccctcc ctattgccac ggcggaactc atcgccgcct gccttgcccgctgctggaca 4980 ggggctcggc tgttgggcac tgacaattcc gtggtgttgt cggggaaatcatcgtccttt 5040 ccttggctgc tcgcctgtgt tgccacctgg attctgcgcg ggacgtccttctgctacgtc 5100 ccttcggccc tcaatccagc ggaccttcct tcccgcggcc tgctgccggctctgcggcct 5160 cttccgcgtc ttcgccttcg ccctcagacg agtcggatct ccctttgggccgcctccccg 5220 catcgatacc gtcgacctcg agacctagaa aaacatggag caatcacaagtagcaataca 5280 gcagctacca atgctgattg tgcctggcta gaagcacaag aggaggaggaggtgggtttt 5340 ccagtcacac ctcaggtacc tttaagacca atgacttaca aggcagctgtagatcttagc 5400 cactttttaa aagaaaaggg gggactggaa gggctaattc actcccaacgaagacaagat 5460 atccttgatc tgtggatcta ccacacacaa ggctacttcc ctgattggcagaactacaca 5520 ccagggccag ggatcagata tccactgacc tttggatggt gctacaagctagtaccagtt 5580 gagcaagaga aggtagaaga agccaatgaa ggagagaaca cccgcttgttacaccctgtg 5640 agcctgcatg ggatggatga cccggagaga gaagtattag agtggaggtttgacagccgc 5700 ctagcatttc atcacatggc ccgagagctg catccggact gtactgggtctctctggtta 5760 gaccagatct gagcctggga gctctctggc taactaggga acccactgcttaagcctcaa 5820 taaagcttgc cttgagtgct tcaagtagtg tgtgcccgtc tgttgtgtgactctggtaac 5880 tagagatccc tcagaccctt ttagtcagtg tggaaaatct ctagcagggcccgtttaaac 5940 ccgctgatca gcctcgactg tgccttctag ttgccagcca tctgttgtttgcccctcccc 6000 cgtgccttcc ttgaccctgg aaggtgccac tcccactgtc ctttcctaataaaatgagga 6060 aattgcatcg cattgtctga gtaggtgtca ttctattctg gggggtggggtggggcagga 6120 cagcaagggg gaggattggg aagacaatag caggcatgct ggggatgcggtgggctctat 6180 ggcttctgag gcggaaagaa ccagctgggg ctctaggggg tatccccacgcgccctgtag 6240 cggcgcatta agcgcggcgg gtgtggtggt tacgcgcagc gtgaccgctacacttgccag 6300 cgccctagcg cccgctcctt tcgctttctt cccttccttt ctcgccacgttcgccggctt 6360 tccccgtcaa gctctaaatc gggggctccc tttagggttc cgatttagtgctttacggca 6420 cctcgacccc aaaaaacttg attagggtga tggttcacgt agtgggccatcgccctgata 6480 gacggttttt cgccctttga cgttggagtc cacgttcttt aatagtggactcttgttcca 6540 aactggaaca acactcaacc ctatctcggt ctattctttt gatttataagggattttgcc 6600 gatttcggcc tattggttaa aaaatgagct gatttaacaa aaatttaacgcgaattaatt 6660 ctgtggaatg tgtgtcagtt agggtgtgga aagtccccag gctccccagcaggcagaagt 6720 atgcaaagca tgcatctcaa ttagtcagca accaggtgtg gaaagtccccaggctcccca 6780 gcaggcagaa gtatgcaaag catgcatctc aattagtcag caaccatagtcccgccccta 6840 actccgccca tcccgcccct aactccgccc agttccgccc attctccgccccatggctga 6900 ctaatttttt ttatttatgc agaggccgag gccgcctctg cctctgagctattccagaag 6960 tagtgaggag gcttttttgg aggcctaggc ttttgcaaaa agctcccgggagcttgtata 7020 tccattttcg gatctgatca gcacgtgttg acaattaatc atcggcatagtatatcggca 7080 tagtataata cgacaaggtg aggaactaaa ccatggccaa gttgaccagtgccgttccgg 7140 tgctcaccgc gcgcgacgtc gccggagcgg tcgagttctg gaccgaccggctcgggttct 7200 cccgggactt cgtggaggac gacttcgccg gtgtggtccg ggacgacgtgaccctgttca 7260 tcagcgcggt ccaggaccag gtggtgccgg acaacaccct ggcctgggtgtgggtgcgcg 7320 gcctggacga gctgtacgcc gagtggtcgg aggtcgtgtc cacgaacttccgggacgcct 7380 ccgggccggc catgaccgag atcggcgagc agccgtgggg gcgggagttcgccctgcgcg 7440 acccggccgg caactgcgtg cacttcgtgg ccgaggagca ggactgacacgtgctacgag 7500 atttcgattc caccgccgcc ttctatgaaa ggttgggctt cggaatcgttttccgggacg 7560 ccggctggat gatcctccag cgcggggatc tcatgctgga gttcttcgcccaccccaact 7620 tgtttattgc agcttataat ggttacaaat aaagcaatag catcacaaatttcacaaata 7680 aagcattttt ttcactgcat tctagttgtg gtttgtccaa actcatcaatgtatcttatc 7740 atgtctgtat accgtcgacc tctagctaga gcttggcgta atcatggtcatagctgtttc 7800 ctgtgtgaaa ttgttatccg ctcacaattc cacacaacat acgagccggaagcataaagt 7860 gtaaagcctg gggtgcctaa tgagtgagct aactcacatt aattgcgttgcgctcactgc 7920 ccgctttcca gtcgggaaac ctgtcgtgcc agctgcatta atgaatcggccaacgcgcgg 7980 ggagaggcgg tttgcgtatt gggcgctctt ccgcttcctc gctcactgactcgctgcgct 8040 cggtcgttcg gctgcggcga gcggtatcag ctcactcaaa ggcggtaatacggttatcca 8100 cagaatcagg ggataacgca ggaaagaaca tgtgagcaaa aggccagcaaaaggccagga 8160 accgtaaaaa ggccgcgttg ctggcgtttt tccataggct ccgcccccctgacgagcatc 8220 acaaaaatcg acgctcaagt cagaggtggc gaaacccgac aggactataaagataccagg 8280 cgtttccccc tggaagctcc ctcgtgcgct ctcctgttcc gaccctgccgcttaccggat 8340 acctgtccgc ctttctccct tcgggaagcg tggcgctttc tcatagctcacgctgtaggt 8400 atctcagttc ggtgtaggtc gttcgctcca agctgggctg tgtgcacgaaccccccgttc 8460 agcccgaccg ctgcgcctta tccggtaact atcgtcttga gtccaacccggtaagacacg 8520 acttatcgcc actggcagca gccactggta acaggattag cagagcgaggtatgtaggcg 8580 gtgctacaga gttcttgaag tggtggccta actacggcta cactagaagaacagtatttg 8640 gtatctgcgc tctgctgaag ccagttacct tcggaaaaag agttggtagctcttgatccg 8700 gcaaacaaac caccgctggt agcggtggtt tttttgtttg caagcagcagattacgcgca 8760 gaaaaaaagg atctcaagaa gatcctttga tcttttctac ggggtctgacgctcagtgga 8820 acgaaaactc acgttaaggg attttggtca tgagattatc aaaaaggatcttcacctaga 8880 tccttttaaa ttaaaaatga agttttaaat caatctaaag tatatatgagtaaacttggt 8940 ctgacagtta ccaatgctta atcagtgagg cacctatctc agcgatctgtctatttcgtt 9000 catccatagt tgcctgactc cccgtcgtgt agataactac gatacgggagggcttaccat 9060 ctggccccag tgctgcaatg ataccgcgag acccacgctc accggctccagatttatcag 9120 caataaacca gccagccgga agggccgagc gcagaagtgg tcctgcaactttatccgcct 9180 ccatccagtc tattaattgt tgccgggaag ctagagtaag tagttcgccagttaatagtt 9240 tgcgcaacgt tgttgccatt gctacaggca tcgtggtgtc acgctcgtcgtttggtatgg 9300 cttcattcag ctccggttcc caacgatcaa ggcgagttac atgatcccccatgttgtgca 9360 aaaaagcggt tagctccttc ggtcctccga tcgttgtcag aagtaagttggccgcagtgt 9420 tatcactcat ggttatggca gcactgcata attctcttac tgtcatgccatccgtaagat 9480 gcttttctgt gactggtgag tactcaacca agtcattctg agaatagtgtatgcggcgac 9540 cgagttgctc ttgcccggcg tcaatacggg ataataccgc gccacatagcagaactttaa 9600 aagtgctcat cattggaaaa cgttcttcgg ggcgaaaact ctcaaggatcttaccgctgt 9660 tgagatccag ttcgatgtaa cccactcgtg cacccaactg atcttcagcatcttttactt 9720 tcaccagcgt ttctgggtga gcaaaaacag gaaggcaaaa tgccgcaaaaaagggaataa 9780 gggcgacacg gaaatgttga atactcatac tcttcctttt tcaatattattgaagcattt 9840 atcagggtta ttgtctcatg agcggataca tatttgaatg tatttagaaaaataaacaaa 9900 taggggttcc gcgcacattt ccccgaaaag tgccacctga c 9941 3 233DNA Homo sapiens 3 gaattcgaac gctgacgtca tcaacccgct ccaaggaatcgcgggcccag tgtcactagg 60 cgggaacacc cagcgcgcgt gcgccctggc aggaagatggctgtgaggga caggggagtg 120 gcgccctgca atatttgcat gtcgctatgt gttctgggaaatcaccataa acgtgaaatg 180 tctttggatt tgggaatctt ataagttctg tatgagaccacagatctaag ctt 233 4 355 DNA Artificial Sequence This represents amutant human sequence having an introduced bacterial tetO1 binding site.4 gggaattccc ccagtggaaa gacgcgcagg caaaacgcac cacgtgacgg agcgtgaccg 60cgcgccgagc ccaaggtcgg gcaggaagag ggcctatttc ccatgattcc ttcatatttg 120catatacgat acaaggctgt tagagagata attagaatta atttgactgt aaacacaaag 180atattagtac aaaatacgtg acgtagaaag taataatttc ttgggtagtt tgcagtttta 240aaattatgtt ttaaaatgga ctatcatatg cttaccgtaa cttgaaagta ctctatcatt 300gatagagtta tatatcttgt ggaaaggacg aaacaccgtg gtcttcaagc ttccg 355 5 634DNA E. coli 5 gctagccacc atgtccagat tagataaaag taaagtgatt aacagcgcattagagctgct 60 taatgaggtc ggaatcgaag gtttaacaac ccgtaaactc gcccagaagctaggtgtaga 120 gcagcctaca ttgtattggc atgtaaaaaa taagcgggct ttgctcgacgccttagccat 180 tgagatgtta gataggcacc atactcactt ttgcccttta gaaggggaaagctggcaaga 240 ttttttacgt aataacgcta aaagttttag atgtgcttta ctaagtcatcgcgatggagc 300 aaaagtacat ttaggtacac ggcctacaga aaaacagtat gaaactctcgaaaatcaatt 360 agccttttta tgccaacaag gtttttcact agagaatgca ttatatgcactcagcgctgt 420 ggggcatttt actttaggtt gcgtattgga agatcaagag catcaagtcgctaaagaaga 480 aagggaaaca cctactactg atagtatgcc gccattatta cgacaagctatcgaattatt 540 tgatcaccaa ggtgcagagc cagccttctt attcggcctt gaattgatcatatgcggatt 600 agaaaaacaa cttaaatgtg aaagtgggtc ttaa 634

What is claimed is:
 1. A method of expressing an RNA molecule within acell, the method comprising: transfecting a packaging cell line with aretroviral construct; recovering recombinant retrovirus from thepackaging cell line; and infecting a target cell with the recombinantretrovirus, wherein the retroviral construct comprises the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR), aself-inactivating lentiviral 3′ LTR, an RNA Polymerase III promoterregion and an RNA coding region operably linked to an RNA Polymerase IIIpromoter region.
 2. The method of claim 1, wherein the retroviralconstruct further comprises at least one termination sequence operablylinked to the RNA coding region.
 3. The method of claim 1, wherein theRNA Polymerase III promoter is inducible.
 4. The method of claim 3,wherein the inducible promoter is activated with tetracycline.
 5. Themethod of claim 1, wherein the RNA coding region encodes aself-complementary RNA molecule having a sense region, an antisenseregion and a loop region.
 6. The method of claim 5, wherein the loopregion is about 2 to about 10 nucleotides in length.
 7. The method ofclaim 5, wherein the sense region and the antisense region are eachbetween about 15 and about 30 nucleotides in length.
 8. The method ofclaim 1, wherein the retroviral construct comprises a first RNA codingregion operably linked to a first RNA Polymerase III promoter and asecond RNA coding region operably linked to a second RNA Polymerase IIIpromoter
 9. The method of claim 8, wherein the first RNA coding regionencodes a first RNA molecule and the second RNA coding region encodes asecond RNA molecule.
 10. The method of claim 9, wherein the first RNAmolecule and the second RNA molecule are substantially complementary.11. The method of claim 1, wherein the retroviral construct comprises afirst RNA Polymerase III promoter and a second RNA Polymerase IIIpromoter, each operably linked to the RNA coding region, such thatexpression of the RNA coding region from the first RNA Polymerase IIIpromoter results in the synthesis of a first RNA molecule and expressionof the RNA coding region from the second RNA Polymerase III promoterresults in the synthesis of a second RNA molecule substantiallycomplementary to the first RNA molecule.
 12. The method of claim 1,wherein expression of the RNA coding region results in the downregulation of a target gene, wherein the target gene comprises asequence that is at least about 90% identical with the RNA codingregion.
 13. The method of claim 1 wherein said packaging cell line is a293 cell line.
 14. The method of claim 1 wherein the 5′ LTR sequencesare from HIV.
 15. The method of claim 1, wherein the viral constructcomprises the woodchuck hepatitis virus enhancer element sequence. 16.The method of claim 1, wherein the viral construct comprises a tRNAamber suppressor sequence.
 17. The method of claim 1 wherein theself-inactivating 3′ LTR comprises a U3 element with a deletion of itsenhancer sequence.
 18. The method of claim 17, wherein theself-inactivating 3′ LTR is a modified HIV 3′ LTR.
 19. The method ofclaim 1, wherein the recombinant retrovirus is pseudotyped.
 20. Themethod of claim 19, wherein the recombinant retrovirus is pseudotypedwith the vesicular stomatitits virus envelope glycoprotein.
 21. Themethod of claim 1, wherein the viral construct further comprises a geneof interest.
 22. The method of claim 21, wherein the viral construct hasa Polymerase II promoter operably linked to the gene of interest. 23.The method of claim 22, wherein the promoter is a CMV promoter.
 24. Themethod of claim 22, wherein the viral construct additionally comprisesan enhancer operably linked to the promoter.
 25. The method of claim 24,wherein the enhancer and promoter are CMV sequences.
 26. The method ofclaim 21, wherein the gene of interest is a reporter gene.
 27. Themethod of claim 26, wherein the reporter gene encodes a fluorescentprotein.
 28. The method of claim 27, wherein the fluorescent protein isgreen fluorescent protein.
 29. The method of claim 20, wherein thepolymerase II promoter is a ubiquitous promoter.
 30. The method of claim27, wherein the ubiquitous promoter is selected from the groupconsisting of the ubiquitin promoter, the CMV β-actin promoter and thepgk promoter.
 31. The method of claim 22, wherein the RNA Polymerase IIpromoter is a tissue specific promoter.
 32. The method of claim 31,wherein said tissue specific promoter is selected from the groupconsisting of the lck promoter, the myogenin promoter and the thylpromoter.
 33. The method of claim 1, wherein the target cell is anembryonic cell.
 34. The method of claim 33, wherein the target cell isan embryogenic stem cell.
 35. The method of claim 33, further comprisingimplanting the embryo in a pseudopregnant female.
 36. The method ofclaim 1, wherein infecting a target cell comprises injecting therecombinant retrovirus between the zona pellucida and the cell membraneof a mammalian embryonic cell.
 37. The method of claim 1, whereininfecting a target cell comprises removing the zona pellucida from amammalian embryonic cell and incubating the cell in solution containingthe recombinant retrovirus.
 38. The method of claim 37, wherein the zonapellucida is removed by enzymatic digestion.
 39. The method of claim 1,wherein the target cell is a cultured cell.
 40. The method of claim 39,wherein the target cell is a cultured mammalian cell.
 41. The method ofclaim 40, wherein the cultured mammalian cell is selected from the groupconsisting of CHO, HEK, COS and MEF cells.
 42. The method of claim 1,wherein the target cell is an embryonic cell of a bird.
 43. The methodof claim 41, wherein infecting an embryonic cell of a bird comprisescontacting the embryonic blastodisc of the bird egg with the recombinantretrovirus.
 44. The method of claim 1, wherein the target cell is a fishegg.
 45. The method of claim 42, wherein infecting the fish eggcomprises delivering the recombinant retrovirus to the space between thechorion and the cell membrane of the fish egg.
 46. A retroviralconstruct for the expression of an RNA molecule within a cell, theretroviral construct comprising: a nucleic acid having the R and U5sequences from a 5′ lentiviral long terminal repeat (LTR); aself-inactivating lentiviral 3′ LTR; and an RNA Polymerase III promoter.47. The retroviral construct of claim 46, further comprising at leastone termination sequence.
 48. The retroviral construct of claim 46,wherein the RNA Polymerase III promoter is inducible.
 49. The retroviralconstruct of claim 48, wherein the inducible promoter is activated withtetracycline.
 50. The retroviral construct of claim 46, furthercomprising at least one RNA coding region operably linked to the RNAPolymerase III promoter.
 51. The retroviral construct of claim 50,wherein the RNA coding region encodes a self-complementary RNA moleculehaving a sense region, an antisense region and a loop region.
 52. Theretroviral construct of claim 51, wherein the loop region is about 2 toabout 10 nucleotides in length.
 53. The retroviral construct of claim51, wherein the sense region and the antisense region are between about15 and about 30 nucleotides in length.
 54. The retroviral construct ofclaim 50, wherein the RNA coding region encodes a first RNA molecule,and the retroviral construct further comprises a second RNA PolymeraseIII promoter and a second RNA coding region operably linked to thesecond RNA Polymerase III promoter, wherein the second RNA coding regionencodes a second RNA molecule substantially complementary to the firstRNA molecule.
 55. The retroviral construct of claim 50, wherein theretroviral construct further comprises a second RNA Polymerase IIIpromoter operably linked to the RNA coding region, such that expressionof the RNA coding region from the first RNA Polymerase III promoterresults in a synthesis of a first RNA molecule and expression of the RNAcoding region from the second RNA Polymerase III promoter results insynthesis of a second RNA molecule substantially complementary to thefirst RNA molecule.
 56. The retroviral construct of claim 50, whereinexpression of the RNA coding region results in the down regulation of atarget gene,.
 57. The method of claim 56, wherein the target genecomprises a sequence that is at least about 90% identical with the RNAcoding region.
 58. The retroviral construct of claim 46, wherein the 5′LTR sequences are from HIV.
 59. The retroviral construct of claim 46,wherein the viral construct comprises the woodchuck hepatitis virusenhancer element sequence.
 60. The retroviral construct of claim 46,wherein the viral construct comprises a tRNA amber suppressor sequence.61. The retroviral construct of claim 46, wherein the self-inactivating3′ LTR comprises a U3 element with a deletion of its enhancer sequence.62. The retroviral construct of claim 46, wherein the self-inactivating3′ LTR is a modified HIV 3′ LTR.
 63. The retroviral construct of claim46, wherein the recombinant retrovirus is pseudotyped.
 64. Theretroviral construct of claim 63, wherein the recombinant retrovirus ispseudotyped with the vesicular stomatitits virus envelope glycoprotein.65. The retroviral construct of claim 46, wherein the viral constructfurther comprises a gene of interest.
 66. The retroviral construct ofclaim 65, wherein the viral construct has a Polymerase II promoteroperably linked to the gene of interest.
 67. The retroviral construct ofclaim 66, wherein the RNA Polymerase II promoter is a CMV promoter. 68.The retroviral construct of claim 66, wherein the viral constructadditionally comprises an enhancer operably linked to the RNA PolymeraseII promoter.
 69. The retroviral construct of claim 68, wherein theenhancer and the RNA Polymerase II promoter are CMV sequences.
 70. Theretroviral construct of claim 66, wherein the RNA Polymerase II promoteris a ubiquitous promoter.
 71. The retroviral construct of claim 70,wherein the ubiquitous promoter is selected from the group consisting ofthe ubiquitin promoter, the CMV β-actin promoter and the pgk promoter.72. The retroviral construct of claim 66, wherein the RNA Polymerase IIpromoter is a tissue specific promoter.
 73. The retroviral construct ofclaim 72, wherein said tissue specific promoter is selected from thegroup consisting of the lck promoter, the myogenin promoter and the thylpromoter.
 74. The retroviral construct of claim 65, wherein the gene ofinterest is a reporter gene.
 75. The retroviral construct of claim 74,wherein the reporter gene encodes a fluorescent protein.
 76. Theretroviral construct of claim 75, wherein the fluorescent protein isgreen fluorescent protein.